Yeast Host Cells and Methods for Producing Fatty Alcohols

ABSTRACT

The present invention provides for a genetically modified yeast cell comprising at least six or more of the following modifications: increased expression of Mus musculus fatty acid reductase, acetyl-CoA carboxylase, fatty acid synthase 1, fatty acid synthase 2, a mutant of the bottleneck enzyme encoded by ACC1 insensitive to post-transcriptional and post-translational repression, and/or a desaturase encoded by OLE1, and reduced expression of DGA1, HFD1, ADH6, and/or GDH1. The present invention provides a method for constructing the genetically modified yeast cell, and a method for producing a fatty alcohol from the genetically modified yeast cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/505,725, filed May 12, 2017, which is herein incorporated byreference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

The invention was made with government support under Contract No.DE-AC02-05CH11231 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is in the field of producing fatty alcohols fromengineered yeast cells.

BACKGROUND OF THE INVENTION

Several laboratories have reported on engineering the fatty acid pathwayin a microbial yeast such as Saccharomyces cerevisiae as a sustainableplatform for producing biofuels and other products (Kalscheuer et al.,2006; Runguphan and Keasling, 2013). A range of enzymes and pathwayshave been heterologously expressed to convert fatty acidthioesters—produced by endogenous fatty acid biosynthesis—into ethylesters, acids, alcohols, alkanes, methyl ketones, dicarboxylic acids,etc. (Clomburg et al., 2015; Goh et al., 2012; Zhou et al., 2016). Amongthese products, long-chain fatty alcohols in the C12-C18 range haverecently received intense attention due to their value and broadapplications in laundry detergents, industrial lubricants andsurfactants, medicines and personal care products, and potentially asbiofuels (Feng et al., 2015; Liu et al., 2016; Pfleger et al., 2015). In2016, the worldwide market for fatty alcohols was $3.7 billion andgrowing, with annual production of more than 2.6 metric tons sourcedprimarily from fossil fuels (petroleum, or polymerized natural gas) orplant oil crops (triglycerides) processed chemically into alcohols(Gaikwad, 2016). Microbial production, besides providing a moresustainable source, can allow for highly specific chemical modifications(Haushalter et al., 2014) to improve product performance or create newapplications in ways that could be difficult or impossible bytraditional thermochemical means.

However, low yields and economic competition from mature petrochemicalprocesses hamper widespread adoption of microbial fatty alcoholproduction. Traditionally, S. cerevisiae is the preferred industrialbiorefinery yeast due to its genomic and structural robustness, andsince existing ethanol-producing fermentation facilities could beretrofitted for another product. Yet yields of fatty alcohols in S.cerevisiae stand at less than 2% of the theoretical maximum from glucose(Zhou et al., 2016). Besides product yield, economic viability alsodepends on the choice of feedstocks. Sugars derived from food crops arecost-prohibitive and divert water and other resources in a period wheredemand for food and water is expected to increase ˜50% by the turn ofthis century (Connor and Uhlenbrook, 2016). Using lignocellulosicfeedstocks derived from agricultural waste or energy crops that do notcompete for water and land with food would lower costs and providemaximal CO2 emission offsets (Caspeta and Nielsen, 2013).

SUMMARY OF THE INVENTION

The present invention provides for a genetically modified yeast cellcomprising at least six or more of the following modifications: (a) anincreased expression of Mus musculus fatty acid reductase (FAR)(MmFAR1), or functional fragment thereof; (b) an increased expression ofacetyl-CoA carboxylase (ACC1), or functional fragment thereof; (c) anincreased expression of fatty acid synthase 1 (FAS1), or functionalfragment thereof; (d) an increased expression of fatty acid synthase 2(FAS2), or functional fragment thereof; (e) a reduced expression of orknocked out for DGA1; (f) a reduced expression of or knocked out forHFD1; (g) a reduced expression of or knocked out for ADH6; (h) anincreased expression of a mutant of the bottleneck enzyme encoded byACC1 insensitive to post-transcriptional and post-translationalrepression, or functional fragment thereof; (i) a reduced expression ofor knocked out for GDH1; and (j) an increased expression of thedesaturase encoded by OLE1, or functional fragment thereof.

In some embodiments, the genetically modified yeast cell comprises atleast seven or more of the modifications. In some embodiments, thegenetically modified yeast cell comprises at least eight or more of themodifications. In some embodiments, the genetically modified yeast cellcomprises at least nine or more of the modifications. In someembodiments, the genetically modified yeast cell comprises at least tenor more of the modifications.

In some embodiments, the yeast cell is a Saccharomyces cell. In someembodiments, the Saccharomyces cell is a Saccharomyces cerevisiae cell.In some embodiments, the Saccharomyces cerevisiae cell is a cell of theSaccharomyces cerevisiae BY4741 strain.

The present invention also provides for a method of constructing thegenetically modified yeast cell of the present invention comprising atleast six or more of the following steps: (a) introducing a firstnucleic acid encoding MmFAR1, or functional fragment thereof,operatively linked to a promoter capable of expressing the MmFAR1 geneproduct in the yeast cell; (b) introducing a second nucleic acidencoding ACC1, or functional fragment thereof, operatively linked to apromoter capable of expressing the ACC1 gene product in the yeast cell,or replacing the native promoter of ACC1 with a promoter with a highertranscription activity, such as promoter TEF1; (c) introducing a thirdnucleic acid encoding FAS1, or functional fragment thereof, operativelylinked to a promoter capable of expressing the FAS1 gene product in theyeast cell, or replacing the native promoter of FAS1 with a promoterwith a higher transcription activity, such as promoter TEF1; (d)introducing a fourth nucleic acid encoding FAS2, or functional fragmentthereof, operatively linked to a promoter capable of expressing the FAS2gene product in the yeast cell or replacing the native promoter of FAS2with a promoter with a higher transcription activity, such as promoterTEF1; (e) removing all or a portion of the DGA1 gene, or introducing anucleic acid into the yeast cell targeting the DGA1 gene or its geneproduct, such that the yeast cell has a reduced amount of activity ofthe DGA1 gene product; (f) removing all or a portion of the HFD1 gene,or introducing a nucleic acid into the yeast cell targeting the HFD1gene or its gene product, such that the yeast cell has a reduced amountof activity of the HFD1 gene product; (g) removing all or a portion ofthe ADH6 gene, or introducing a nucleic acid into the yeast celltargeting the ADH6 gene or its gene product, such that the yeast cellhas a reduced amount of activity of the ADH6 gene product; (h)introducing a fifth nucleic acid encoding a mutant of the bottleneckenzyme encoded by ACC1 insensitive to post-transcriptional andpost-translational repression, or functional fragment thereof,operatively linked to a promoter capable of expressing the mutant ACC1gene product in the yeast cell; (i) removing all or a portion of theGDH1 gene, or introducing a nucleic acid into the yeast cell targetingthe GDH1 gene or its gene product, such that the yeast cell has areduced amount of activity of the GDH1 gene product; (j) introducing asixth nucleic acid encoding OLE1, or functional fragment thereof,operatively linked to a promoter capable of expressing the OLE1 geneproduct in the yeast cell; and (k) introducing a seventh nucleic acidencoding a second copy of MmFAR1, or functional fragment thereof,operatively linked to a promoter capable of expressing the MmFAR1 geneproduct in the yeast cell. In some embodiments, alternatively theintroducing steps of (e), (f), (g), and/or (i) independently compriseintroducing gRNA into the yeast cell, such as using dCAS9, to inhibiteach respective gene throuogh gene silencing or antisense or the like.

In some embodiments, two or more of the one to six nucleic acids of themethod reside on a single nucleic acid. In some embodiments, the nucleicacid is capable of stable maintenance in the genetically modified yeastcell. In some embodiments, the nucleic acid is a vector capable ofstable maintenance in the genetically modified yeast cell. In someembodiments, the nucleic acid introduced into the genetically modifiedyeast cell causes the stable integration of the nucleic acid in achromosome of the genetically modified yeast cell. In some embodiments,the OLE1 is a fatty acid-desaturase encoded by OLE1.

The present invention further provides for a method of producing a fattyalcohol from a genetically modified yeast cell comprising: (a) providinga genetically modified yeast cell of the present invention, and (b)growing or culturing the genetically modified yeast cell in a mediumsuch that the genetically modified yeast cell produces one or more fattyalcohols, or a mixture thereof.

In some embodiments, the method further comprises one or more steps ofthe method of constructing the genetically modified yeast cell of thepresent invention. In some embodiments, each one liter of thegenetically modified yeast cell grown or cultured in the medium produces0.2, 0.4, 0.6, 0.8, or 1.0 g or more of fatty alcohol. In someembodiments, the growing or culturing step (b) comprises growing orculturing the genetically modified yeast cell in a fed-batch orcontinuous culture. In some embodiments, each one liter of thegenetically modified yeast cell grown or cultured in the fed-batch orcontinuous culture produces 1.0, 2.0, 3.0, 4.0, 5.0, or 6.0 g or more offatty alcohol. A production of 6.0 g/L of fatty alcohol corresponds toabout 20% of the maximum theoretical yield from glucose.

In some embodiments, the medium comprises a carbon source produced froma biomass, such as a lignocellulosic feedstock. In some embodiments, thecarbon source produced from the biomass is obtained by deconstructing anon-food crop using a cholinium-based renewable ionic liquid (IL). Insome embodiments, the medium is fed a constant from about 1.5 g/hr toabout 3.0 g/hr glucose, or pulse-fed about 1 g/hr glucose.

Four heterologous fatty acid reductases are compared and activity and ERlocalization is found high using a Mus musculus FAR. From screening anadditional 21 single-gene edits, the following successful strategies toimprove titer are identified: a strain containing eleven geneticmodifications compared to the parent BY4741 strain produced 1.2 g/Lfatty alcohols in shake flasks.

High-level production from feedstocks produced from non-food crops andcholinium-based renewable bionic ILs reaching a titer of 0.7 g/L inshake flasks are demonstrated. Scale-up fermentation and exploringalternative feeding strategies aimed at limiting overflow metabolism,achieving a titer of 6.0 g/L in a 2-L, fed-batch bioreactor, aredemonstrated. These titers are the highest for fatty alcohols reportedto date for S. cerevisiae. This is the first report of a bioproductproduced by yeast from feedstocks derived solely from biomass.

In some embodiments, the fatty alcohol is a fatty alcohol in the C12-C18range. Fatty alcohols in the C12-C18 range are used in personal careproducts, lubricants, and potentially biofuels.

The present invention relates to genetic modifications to yeast toproduce high levels of long-chain alcohols in the C12-C18 rangecontaining terminal alcohol groups. The production levels are in the1-10 g/L range and are the highest to date in engineered Saccharomycescerevisiae. This invention also relates to high-level production usingfeedstocks derived from lignocellulose, such as, sorghum or switchgrass.This invention relates to using lignocellulosic biomass pre-treated withionic liquids as the sole carbon source for fatty alcohol production bythe engineered microorganisms. The technical problems overcome toachieve this high-level production are screening dozens of geneticmodifications to find changes that led to increased production and/orgrowth of the engineered strains. These genetic changes includescreening heterologous fatty acid reductase (FAR) enzymes and findinghighest activity and endoplasmic reticulum localization from a Musmusculus FAR. In some embodiments, the genetically modified yeast cellcomprises an increased FAR expression; deleting competing reactionsencoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoAcarboxylase; limiting NADPH and carbon usage by the glutamatedehydrogenase encoded by GDH1; and overexpressing the fattyacid-desaturase encoded by OLE1. In some embodiments, the geneticallymodified yeast cell produces 1.0, 1.1, 1.2, or 1.3 g/L or more fattyalcohols in shake flasks, and 6.0 g/L in fed-batch fermentation,corresponding to about 20% of the maximum theoretical yield fromglucose, the highest titers and yields reported to date in S.cerevisiae. In some embodiments, the genetically modified yeast cell isgrown using a carbon source produced from lignocellulosic feedstocksderived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks.

Fatty alcohols can be used as industrial lubricants, in personal careproducts (e.g., conditioners, creams, lotions, shampoos), and asbiofuels. Fatty alcohols produced from microbial cell factories fromlignocellulosic biomass are renewable. Alternative petroleum-basedsourcing is not renewable and oil crops compete with food for land andwater. Lignocellulosic feedstocks (e.g., switchgrass, sorghum) can beconverted to fatty alcohols at high titer through the present inventionand provide a green sourcing for these important products.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and others will be readily appreciated by theskilled artisan from the following description of illustrativeembodiments when read in conjunction with the accompanying drawings.

FIG. 1. Schema for fatty chemical engineering showing metabolic flux andprotein levels of previous first-generation producer. The highest-titerchemical producer strain (yL401) from our previous study (Runguphan andKeasling, 2013) was analyzed by shotgun proteomics (Batth et al., 2012)and metabolic flux analysis (García Martín et al., 2015) to identifytargets for engineering increased production. Carbon flux is indicatedby orange lines, the width proportional to net normalized flux throughthe corresponding reaction. Fluxes of reducing equivalents (from NADH orNADPH) are indicated by blue lines (with the thickness proportional tomolar flux, which is not on the same scale as carbon flux). Fluxes toCO₂, biomass, and minor products are not shown. Enzyme levels areindicated by the areas of the red circles. The genes underlined areoverexpressed in the first-generation strain. *Fatty acid reductase(FAR) is used to produce fatty alcohols from fatty acyl-CoA produced byfatty acid biosynthesis, with the three fatty acid biosynthetic genesoverexpressed in the first-generation producer strain. The flux andproteomics analysis is performed on a free fatty acid producer,containing a thioesterase rather than a FAR.

FIG. 2A. Comparing four heterologous fatty acid reductases in S.cerevisiae. Four FARs are chromosomally integrated into a fatty acyl-CoAoverproducing strain (WRY1) and fatty alcohol production by theresulting strains is compared. Abbrev.: MaFAR2, Maqu2220 fromMarinobacter aquaeoli; MaFAR7, Maqu2507 from the same organism; TaFAR1,FAR1 from Tyto alba; MmFAR1, FAR1 from Mus musculus. All four FARs areexpressed from the same 208a locus, TEF1 promoter, and CYC/terminator.Cultures are grown in 5 ml YPD overlaid with 10% dodecane for threedays. The bars represent the mean, and the error bars one standarddeviation, for three to four biological replicates.

FIG. 2B. Comparing four heterologous fatty acid reductases in S.cerevisiae. Confocal microscopy images of yL405 containing MmFAR-GFPshows the fusion enzyme to be ER-localized, as are many native enzymesthat use the same substrate fatty acyl-CoA (Natter et al., 2005).

FIG. 3A. Genetic modifications to improve fatty alcohol production.Genetic modifications to yL405 (an acyl-CoA overproducing straincontaining one copy of the best-performing MmFAR) are screened inparallel for improvements to fatty alcohol production, with titer shownas a fraction of the parent yL405. Culture is grown in plastic 24-wellplates containing 1 ml YPG overlaid with 20% dodecane for three days.The bars represent the mean, and the error bars one standard deviation,for three to four biological replicates.

FIG. 3B. Genetic modifications to improve fatty alcohol production.Stacking beneficial single-gene edits leads to additional improvementsin titer culminating in the highest producing strain yL434. Culture isgrown in plastic 24-well plates containing 1 ml YPG overlaid with 20%dodecane for three days. The bars represent the mean, and the error barsone standard deviation, for three to four biological replicates.

FIG. 3C. Genetic modifications to improve fatty alcohol production. Thehigh production level from yL434 results in precipitation of fattyalcohols from the saturated dodecane overlay. The culture is grown in a250-ml baffled flask containing 10 ml YPD overlaid with 20% dodecane forthree days.

FIG. 4A. Fatty alcohol production from lignocellulosic biomass. Biomasspretreatment with ionic liquids (ILs) containing cholinium cation pairedwith bio-based anions, e.g., aspartate, break down lignocellulose to bedepolymerized using cellulase enzymes to free constituent sugars.

FIG. 4B. Fatty alcohol production from lignocellulosic biomass. Thehighest producing yL434 strain grown in biomass hydrolysates media(YPBiomass) or conventional rich (YPD or YPG) media produces high levelsof fatty alcohols. Cultures are grown in plastic 250-ml baffled flaskscontaining 10 ml medium overlaid with 20% dodecane for three days. Thebars represent the mean, and the error bars one standard deviation, forthree replicates.

FIG. 4C. Fatty alcohol production from lignocellulosic biomass. Thehighest producing yL434 strain grown in biomass hydrolysates media(YPBiomass) or conventional rich (YPD or YPG) media produces high yieldsof fatty alcohols. Cultures are grown in plastic 250-ml baffled flaskscontaining 10 ml medium overlaid with 20% dodecane for three days. Thebars represent the mean, and the error bars one standard deviation, forthree replicates.

FIG. 5A. High-density, fed-batch bioreactor fermentations. The highestproducing yL434 strain is grown in 2 L-scale bioreactors under differentfed-batch conditions to maximize titer. Bioreactor A contained half thenormal glucose concentration in batch phase (1% vs. 2% for others) andis fed a constant ˜1.5 g/hr concentrated glucose.

FIG. 5B. High-density, fed-batch bioreactor fermentations. The highestproducing yL434 strain is grown in 2 L-scale bioreactors under differentfed-batch conditions to maximize titer. Bioreactor B is pulse-fed ˜1g/hr (1 g pulses of concentrated glucose upon carbon source depletion asindicated by O₂ spike, occurring every ˜1 hr).

FIG. 5C. High-density, fed-batch bioreactor fermentations. The highestproducing yL434 strain is grown in 2 L-scale bioreactors under differentfed-batch conditions to maximize titer. Bioreactor C is fed a constant˜3 g/hr.

FIG. 5D. High-density, fed-batch bioreactor fermentations. The highestproducing yL434 strain is grown in 2 L-scale bioreactors under differentfed-batch conditions to maximize titer. Bioreactor D is fed a constant˜1.5 g/hr.

DETAILED DESCRIPTION OF THE INVENTION

Before the invention is described in detail, it is to be understoodthat, unless otherwise indicated, this invention is not limited toparticular sequences, expression vectors, enzymes, yeast microorganisms,or processes, as such may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “cell” includes a single cell as well as aplurality of cells; and the like.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terms “optional” or “optionally” as used herein mean that thesubsequently described feature or structure may or may not be present,or that the subsequently described event or circumstance may or may notoccur, and that the description includes instances where a particularfeature or structure is present and instances where the feature orstructure is absent, or instances where the event or circumstance occursand instances where it does not.

The term “about” refers to a value including 10% more than the statedvalue and 10% less than the stated value.

The following abbreviations are used herein:

-   Ac-CoA Acetyl-Coenzyme A, cytoplasmic-   AcaI Acetaldehyde-   ACC1 Acetyl-CoA carboxylase (overexpressed)-   ACC1** ACC1 with two mutations abolishing post-translational    phosphorylation inhibition-   Ace Acetate-   ADH1 Alcohol dehydrogenase (ethanol-forming, NAD-dependent)-   ADH6 Medium chain alcohol dehydrogenase-   ALD6 Aldehyde dehydrogenase (cystolic, NADP+-dependnet)-   ARE1 Acyl-CoA:sterol acyltransferase-   ARE2 Acyl-CoA:sterol acyltransferase-   BcGapN B. cereus Glyceraldehyde-3-phosphate dehydrogenase    (Non-phosphorylating, NADP-dependent)-   DGA1 Diacylglycerol acyltransferase-   FA-CoA Fatty acyl-CoA-   FAL Fatty aldehyde-   FAR Fatty acid reductase (Fatty acyl-CoA reductase, NADPH-dependent)-   FAS 1 Fatty acid synthase (subunit beta)-   FAS2 Fatty acid synthase (subunit alpha)-   FFA Free fatty acid-   G3P Glyceraldehyde 3-phosphate-   G6P Glucose 6-phosphate-   GDH1 Glutamate dehydrogenase (NADPH-dependent)-   GFP A. victoria Green fluorescent protein-   HFA1 cvt Mitochondrial acetyl-CoA carboxylase with mitochondrial    targeting signal deleted-   HFD1 Fatty aldehyde dehydrogenase-   INO2 Transcription factor, derepression of phospholipid biosynthetic    genes-   KlGapDH K. lactis Glyceraldehyde-3-phosphate dehydrogenase    (NADP-dependent)-   LRO1 Diacylglycerol acyltransferase-   Mal-CoA Malonyl-CoA-   MBP E. coli maltose-binding protein-   NADH Nicotinamide adenine dinucleotide (reduced form)-   NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)-   OLE1 Delta(9) fatty acid desaturase-   OL11 Transcription factor, negative regulation of phospholipid    biosynthetic genes-   PGI1 Phosphoglucose isomerase-   PDX1 Fatty-acid coenzyme A oxidase-   PXA1 Peroxisomal fatty acyl-CoA importer (with PXA2)-   Pvr Pyruvate-   RPD3 Histone deacetylase-   SE Steryl esters-   TAG Triacylglycerides-   TCA Tricarboxylic acid cycle-   TDH3 Glyceraldehyde-3-phosphate dehydrogenase (NAD-dependent)-   ZWF1 Glucose-6-phosphate dehydrogenase (NADP-dependent)

The term “heterologous DNA” as used herein refers to a polymer ofnucleic acids wherein at least one of the following is true: (a) thesequence of nucleic acids is foreign to (i.e., not naturally found in) agiven yeast microorganism; (b) the sequence may be naturally found in agiven yeast microorganism, but in an unnatural (e.g., greater thanexpected) amount; or (c) the sequence of nucleic acids comprises two ormore subsequences that are not found in the same relationship to eachother in nature. For example, regarding instance (c), a heterologousnucleic acid sequence that is recombinantly produced will have two ormore sequences from unrelated genes arranged to make a new functionalnucleic acid. Specifically, the present invention describes theintroduction of an expression vector into a yeast microorganism, whereinthe expression vector contains a nucleic acid sequence coding for anenzyme that is not normally found in a yeast microorganism. Withreference to the yeast microorganism's genome, then, the nucleic acidsequence that codes for the enzyme is heterologous.

The terms “expression vector” or “vector” refer to a compound and/orcomposition that transduces, transforms, or infects a yeastmicroorganism, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell, or in a manner not nativeto the cell. An “expression vector” contains a sequence of nucleic acids(ordinarily RNA or DNA) to be expressed by the yeast microorganism.Optionally, the expression vector also comprises materials to aid inachieving entry of the nucleic acid into the yeast microorganism, suchas a virus, liposome, protein coating, or the like. The expressionvectors contemplated for use in the present invention include those intowhich a nucleic acid sequence can be inserted, along with any preferredor required operational elements. Further, the expression vector must beone that can be transferred into a yeast microorganism and replicatedtherein. Preferred expression vectors are plasmids, particularly thosewith restriction sites that have been well documented and that containthe operational elements preferred or required for transcription of thenucleic acid sequence. Such plasmids, as well as other expressionvectors, are well known to those of ordinary skill in the art.

The term “transduce” as used herein refers to the transfer of a sequenceof nucleic acids into a yeast microorganism or cell. Only when thesequence of nucleic acids becomes stably replicated by the cell does theyeast microorganism or cell become “transformed.” As will be appreciatedby those of ordinary skill in the art, “transformation” may take placeeither by incorporation of the sequence of nucleic acids into thecellular genome, i.e., chromosomal integration, or by extrachromosomalintegration. In contrast, an expression vector, e.g., a virus, is“infective” when it transduces a yeast microorganism, replicates, and(without the benefit of any complementary virus or vector) spreadsprogeny expression vectors, e.g., viruses, of the same type as theoriginal transducing expression vector to other microorganisms, whereinthe progeny expression vectors possess the same ability to reproduce.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free of components that normally accompanyit in its native state or free of components from a yeast cell orculture medium from which the material is obtained.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter) and asecond nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

The term “functional fragment” refers to an enzyme that has an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99%identical to the amino acid sequence of any one of the enzymes describedin this specification or in an incorporated reference. The functionalfragment retains amino acids residues that are recognized as conservedfor the enzyme. The functional fragment may have non-conserved aminoacid residues replaced or found to be of a different amino acid, oramino acid(s) inserted or deleted, but which does not affect or hasinsignificant effect on the enzymatic activity of the functionalfragment. The functional fragment has an enzymatic activity that isidentical or essentially identical to the enzymatic activity any one ofthe enzymes described in this specification or in an incorporatedreference. The functional fragment may be found in nature or be anengineered mutant thereof. The mutant may have one or more amino acidssubstituted, deleted or inserted, or a combination thereof, as comparedto the enzyme described in this specification or in an incorporatedreference.

The amino acid sequence of Mus musculus fatty acid reductase (FAR) is asfollows:

(SEQ ID NO: 1)         10         20         30         40MVSIPEYYEG KNILLTGATG FLGKVLLEKL LRSCPRVNSV        50         60         70         80YVLVRQKAGQ TPQERVEEIL SSKLFDRLRD ENPDFREKII        90        100        110        120AINSELTQPK LALSEEDKEI IIDSTNVIFH CAATVRFNEN       130        140        150        160LRDAVQLNVI ATRQLILLAQ QMKNLEVFMH VSTAYAYCNR       170        180        190        200KHIDEVVYPP PVDPKKLIDS LEWMDDGLVN DITPKLIGDR       210        220        230        240PNTYIYTKAL AEYVVQQEGA KLNVAIVRPS IVGASWKEPF       250        260        270        280PGWIDNFNGP SGLFIAAGKG ILRTMRASNN ALADLVPVDV       290        300        310        320VVNTSLAAAW YSGVNRPRNI MVYNCTTGST NPFHWGEVEY       330        340        350        360HVISTFKRNP LEQAFRRPNV NLTSNHLLYH YWIAVSHKAP       370        380        390        400AFLYDIYLRM TGRSPRMMKT ITRLHKAMVF LEYFTSNSWV       410        420        430        440WNTDNVNMLM NQLNPEDKKT FNIDVRQLHW AEYIENYCMG       450        460        470        480 TKKYVLNEEM SGLPAARKHL NKLRNIRYGF NTILVILIWR       490        500        510 IFIARSQMAR NIWYFVVSLC YKFLSYFRAS STMRY

In some embodiments, the yeast cell in its unmodified form has a nativeenzyme of one of the enzymes described herein. In some embodiments, thegene encoding the enzyme of one of the enzymes described herein isdeleted or modified such that expression of the gene is reduced oreliminated. In some embodiments, the yeast cell has a reduced capabilityto catabolize, metabolize, or modify the fatty alcohol.

The nucleic acid constructs of the present invention comprise nucleicacid sequences encoding one or more of the subject enzymes. The nucleicacid of the subject enzymes are operably linked to promoters andoptionally control sequences such that the subject enzymes are expressedin a yeast cell cultured under suitable conditions. The promoters andcontrol sequences are specific for each yeast cell species. In someembodiments, expression vectors comprise the nucleic acid constructs.Methods for designing and making nucleic acid constructs and expressionvectors are well known to those skilled in the art.

Sequences of nucleic acids encoding the subject enzymes are prepared byany suitable method known to those of ordinary skill in the art,including, for example, direct chemical synthesis or cloning. For directchemical synthesis, formation of a polymer of nucleic acids typicallyinvolves sequential addition of 3′-blocked and 5′-blocked nucleotidemonomers to the terminal 5′-hydroxyl group of a growing nucleotidechain, wherein each addition is effected by nucleophilic attack of theterminal 5′-hydroxyl group of the growing chain on the 3′-position ofthe added monomer, which is typically a phosphorus derivative, such as aphosphotriester, phosphoramidite, or the like. Such methodology is knownto those of ordinary skill in the art and is described in the pertinenttexts and literature (e.g., in Matteuci et al. (1980) Tet. Lett.521:719; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637). Inaddition, the desired sequences may be isolated from natural sources bysplitting DNA using appropriate restriction enzymes, separating thefragments using gel electrophoresis, and thereafter, recovering thedesired nucleic acid sequence from the gel via techniques known to thoseof ordinary skill in the art, such as utilization of polymerase chainreactions (PCR; e.g., U.S. Pat. No. 4,683,195).

Each nucleic acid sequence encoding the desired subject enzyme can beincorporated into an expression vector. Incorporation of the individualnucleic acid sequences may be accomplished through known methods thatinclude, for example, the use of restriction enzymes (such as BamHI,EcoRI, HhaI, Xhol, XmaI, and so forth) to cleave specific sites in theexpression vector, e.g., plasmid. The restriction enzyme produces singlestranded ends that may be annealed to a nucleic acid sequence having, orsynthesized to have, a terminus with a sequence complementary to theends of the cleaved expression vector. Annealing is performed using anappropriate enzyme, e.g., DNA ligase. As will be appreciated by those ofordinary skill in the art, both the expression vector and the desirednucleic acid sequence are often cleaved with the same restrictionenzyme, thereby assuring that the ends of the expression vector and theends of the nucleic acid sequence are complementary to each other. Inaddition, DNA linkers may be used to facilitate linking of nucleic acidssequences into an expression vector.

A series of individual nucleic acid sequences can also be combined byutilizing methods that are known to those having ordinary skill in theart (e.g., U.S. Pat. No. 4,683,195).

For example, each of the desired nucleic acid sequences can be initiallygenerated in a separate PCR. Thereafter, specific primers are designedsuch that the ends of the PCR products contain complementary sequences.When the PCR products are mixed, denatured, and reannealed, the strandshaving the matching sequences at their 3′ ends overlap and can act asprimers for each other. Extension of this overlap by DNA polymeraseproduces a molecule in which the original sequences are “spliced”together. In this way, a series of individual nucleic acid sequences maybe “spliced” together and subsequently transduced into a yeastmicroorganism simultaneously. Thus, expression of each of the pluralityof nucleic acid sequences is effected.

In some embodiments, the yeast cells of the present invention aregenetically modified in that heterologous nucleic acid have beenintroduced into the yeast cells, and as such the genetically modifiedyeast cells do not occur in nature. The suitable yeast cell is onecapable of expressing a nucleic acid construct encoding the enzyme(s)described herein. The gene encoding the enzyme may be heterologous tothe yeast cell or the gene may be native to the yeast cell but isoperatively linked to a heterologous promoter and one or more controlregions which result in a higher expression of the gene in the yeastcell. Each enzyme described herein can be native or heterologous to theyeast cell. Where the enzyme is native to the yeast cell, the yeast cellis genetically modified to modulate expression of the enzyme. Thismodification can involve the modification of the chromosomal geneencoding the enzyme in the yeast cell or a nucleic acid constructencoding the gene of the enzyme is introduced into the yeast cell. Oneof the effects of the modification is the expression of the enzyme ismodulated in the yeast cell, such as the increased expression of theenzyme in the yeast cell as compared to the expression of the enzyme inan unmodified yeast cell.

It is to be understood that, while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications withinthe scope of the invention will be apparent to those skilled in the artto which the invention pertains.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

The invention having been described, the following examples are offeredto illustrate the subject invention by way of illustration, not by wayof limitation.

EXAMPLE 1 Engineering High-Level Production of Fatty Alcohols bySaccharomyces cerevisiae from Lignocellulosic Feedstocks

Fatty alcohols in the C12-C18 range are used in personal care products,lubricants, and potentially biofuels. These compounds can be producedfrom the fatty acid pathway by a fatty acid reductase (FAR), yet yieldsfrom the preferred industrial yeast Saccharomyces cerevisiae remainunder 2% of the theoretical maximum from glucose. Titer and yield offatty alcohols are improved using an approach involving quantitativeanalysis of protein levels and metabolic flux, engineering enzyme leveland localization, pull-push-block engineering of carbon flux, andcofactor balancing. Four heterologous FARs are compared, finding highestactivity and endoplasmic reticulum localization from a Mus musculus FAR.After screening an additional twenty-one single-gene edits, increasingFAR expression is identified; deleting competing reactions encoded byDGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase;limiting NADPH and carbon usage by the glutamate dehydrogenase encodedby GDH1; and overexpressing the fatty acid-desaturase encoded by OLE1 assuccessful strategies to improve titer. The final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batchfermentation, corresponding to ˜20% of the maximum theoretical yieldfrom glucose, the highest titers and yields reported to date in S.cerevisiae. High-level production from lignocellulosic feedstocksderived from ionic-liquid treated switchgrass and sorghum isdemostrated, reaching 0.7 g/L in shake flasks. Altogether, these resultsrepresent progress towards efficient and renewable microbial productionof fatty acid-derived products.

Titers and yields of fatty alcohols produced by S. cerevisiae areimproved and demonstrate high-level production from lignocellulosicbiomass hydrolysates. This approach included quantitative analysis ofmetabolic flux and global protein expression to identify pathwaybottlenecks in a first-generation fatty chemical producer strain andidentify genetic modifications for maximizing pathway flux. Among thesemodifications, four heterologous fatty acid reductases—which convertfatty acyl-CoA into fatty alcohols—are compared exploring enzymeexpression level and subcellular localization. Additionally, over twodozen genetic modifications for improvements in fatty alcohol productionare screened and combined beneficial changes into a strain that producedfatty alcohols at high titers and yields. To develop an efficientbioprocess with this strain, fatty alcohol production fromlignocellulosic feedstocks is evaluated in a one-pot process usingcholinium-based ionic liquids. Lastly, successful scale-up in 2 Lfed-batch bioreactors combined with an initial exploration ofalternative substrate feeding strategies is demonstrated to maximizetiter and yield.

Materials and Methods

Generating Strains

All yeast strains in this study are derived from Saccharomycescerevisiae BY4741 (Brachmann et al., 1998), to which the nativepromoters driving ACC1, FAS1, and FAS2 are replaced with the TEF1promoter as previously reported, generating strain WRY1 (Runguphan andKeasling, 2013). All subsequent strains (Table 1) are created viaCas9-aided homologous recombination using the software tool CASdesigner(the webpage of casdesigner.jbei.org) to design integration cassettesand following a previously reported, cloning-free methodology (ReiderApel et al., 2016). Briefly, integration cassettes containing 1-kbflanking homology regions targeting a chosen genomic locus areconstructed by PCR-amplifying donor DNA fragments using primersgenerated by CASdesigner, then co-transformed with a Cas9-gRNA plasmid(pCut) targeting the chosen genomic locus. CASdesigner primers provide30-60 nt of inter-fragment homology allowing 1-5 separate fragments toassemble via homologous recombination in vivo. pCuts targeting emptygenomic loci (e.g., 208a, 1622b) are available pre-cloned, and pCutstargeting new sites (e.g., for deletions) are assembled in vivo from alinear backbone and a linear PCR fragment containing the new gRNAsequence, as described previously (Reider Apel et al., 2016). New gRNAsequences are chosen using DNA2.0 (the webpage ofdna20.com/eCommerce/cas9/input) or CHOP-CHOP (the webpage ofchopchop.rc.fas.harvard.edu) (Montague et al., 2014). To generate donorDNA fragments, native sequences—e.g., chromosomal homology regions,promoters—are amplified from BY4741 genomic DNA, while heterologoussequences—e.g., fatty acid reductase coding sequences—are amplified fromsynthetic gene blocks codon-optimized (for expression in S. cerevisiae)and synthesized by Integrated DNA Technologies (the webpage ofidtdna.com). All PCRs used Phusion Hot Start II DNA polymerase (thewebpage of thermofisher.com, cat. F549L).

Transformations are performed via heat-shock (Gietz and Woods, 2002)using ˜200 ng pCut, ˜1 ug donor DNA per sample, and 20 min heat shock.For assembling a pCut targeting a new site by homologous recombination,200 ng linear pCut backbone and 500 ng of a 1-kb fragment containing thegRNA sequence are used, as described (Reider Apel et al., 2016). Formulti-site integrations, 200 ng total linear pCut backbone, and the sameamounts of gRNA fragment and donor DNA for each site are used as onewould have for a single integration. Colonies are screened by PCRdirected at the target locus, and for integrations, one representativecolony sequenced. Three to four biological replicates are analyzed foreach strain.

Media and Culture Conditions

Selective agar plates used for transformations are purchased fromTeknova (the webpage for: teknova.com, cat. C3080). Liquid selectivemedium used to grow transformants contains 0.2% (w/v) completesupplement mixture (CSM) lacking uracil, 0.67% yeast nitrogen base, and2% dextrose. Nonselective medium contains 1% yeast extract, 2% peptone(Difco cat. 288620 and 211677, respectively), and either 2% dextrose(YPD) or 2% galactose and 0.2% dextrose (YPG). Nonselective agar YPDplates are purchased from Teknova (cat. Y100). Cultures are grown inplastic 96-deep well plates, 24-deep well plastic plates (CWR cat.89080-534), glass test tubes, or 250-ml baffled flasks, as indicated inthe results section. Production cultures are overlaid with dodecane, thelatter spiked with 200 mg/L methyl nonadecanoate (sigma cat. N5377) asan internal standard. Plastic plates are covered with aeraseal film andshaken at 800 rpm in a Multitron shaker. Glass tubes and baffled flasksare shaken at 200 rpm. All strains are grown at 30° C.

Shotgun Proteomics

Strains for proteomics analysis are grown in YPD overnight, thenback-diluted 1:100 into 5 ml fresh YPD and grown 8 hrs. For each sample,the entire culture volume is centrifuged at 3000×g on a table-topcentrifuge, decanted, and the pellet flash-frozen in liquid nitrogen andstored at −80° C. until preparation as described (Batth et al., 2012).Briefly, cell pellets are lysed in urea and bead-beaten. The lysate isreduced using tris(2-carboxyethyl)phosphine (sigma cat. C4706), thenalkylated using iodoacetamide (sigma cat. 11149), trypsinized, desaltedon spin columns, and finally suspended in a buffer of 0.1% formic acidto a final concentration of 2 μg protein/∥l. Peptide data are acquiredusing an Agilent 1290 liquid chromatography system coupled to an Agilent6550 QTOF mass spectrometer and analyzed using Agilent MassHunterversion B.06.00. Resultant data files are searched with Mascot version2.3.02 (the webpage of matrixscience.com) then filtered and validatedusing Scaffold version 4.4.0 (the webpage of proteomesoftware.com).

Metabolic Flux Analysis using ¹³C-Labeled Glucose

Metabolic flux analysis is performed as previously reported (GarcíaMartín et al., 2015; Ghosh et al., 2016). Briefly, strains are grown in250-ml shake flasks in 25 ml medium containing ¹³C-labeled glucose(sigma cat. 407-622-22-9 and 110187-42-3) and sampled at exponentialphase near OD 1.0. Sampling included filtering media forhigh-performance liquid chromatography (HPLC) to quantify extracellularmetabolites, ethyl acetate extraction followed by gaschromatography-mass spectrometry (GC-MS) to quantify fatty acidproducts, and methanol/chloroform extraction followed by liquidchromatography-mass spectrometry (LC-MS/MS) to analyze ¹³C labeling inmetabolites. These ¹³C labeling data are used to constrain the S.cerevisiae genome-scale model iMM904 (Mo et al., 2009) using two-scale¹³C Metabolic Flux Analysis (García Martín et al., 2015; Ghosh et al.,2016) with the open-source, python-based JBEI Quantitative MetabolicModeling library (the webpage of github.com/JBEI/jqmm) to model themetabolic flux distribution.

Fatty Alcohol Production Cultivations

Initial production cultivations are performed as described (Runguphanand Keasling, 2013). Briefly, three to four biological replicates areeach inoculated into 5 ml medium in glass test tubes overnight, thenback-diluted 1:100 into 5 ml of the same medium, overlaid with 0.5 mldodecane (spiked with internal standard), and shaken at 200 rpm forthree days. The overlay is collected and centrifuged at 3000×g, and 10μl of the top organic phase added to 90 μl ethyl acetate in a GC-MS vialcontaining a glass insert for subsequent analysis. This method ofcultivation and sampling is used to compare the fatty acid reductases,in YPD, and for initial cultivations of strain yL405 in biomasshydrolysates and other media.

To screen genetically edited strains for improvements in fatty alcoholproduction, the appearance of precipitated fatty alcohols in somestrains (FIG. 3C) prevented sampling the liquid overlay. Instead, fourbiological replicates are inoculated into 1 ml YPG in 24-deep wellplates and grown overnight, then back-diluted 1:100 into 1 ml YPG,overlaid with 0.2 ml dodecane (spiked with internal standard), coveredwith aeraseal, and shaken at 800 rpm for three days. Then, 100 μl ofculture is added to 800 μl ethyl acetate in 1.6-ml eppendorf tubes,vortexed at maximum setting 30 min, centrifuged at 10,000×g on atable-top centrifuge, and 100 μl of the top organic phase added to aGC-MS vial containing a glass insert for analysis. This method ofcultivation and sampling is used to compare strain yL405 to allsubsequent genetically modified strains.

To characterize production of the final highest-producer strain yL434,four biological replicates are grown in 1 ml corresponding mediumovernight in glass test tubes, then back-diluted 1:100 into 10 ml of thesame medium in 250-ml baffled flasks, overlaid with 2 ml dodecane(spiked with internal standard), and shaken at 200 rpm for three days.Then, 100 μl culture is extracted with ethyl acetate and processed intoGC-MS vials as described immediately above. This method of cultivationand sampling is used to compare production of yL434 in YPG, YPD, andYPBiomass media (see Preparation of biomass hydrolysates).

Fed Batch 2 L-Scale Bioreactor Fermentations

Fed-batch fermentations are performed in Sartorius 2 L bioreactorsequipped Sartorius BIOSTAT B Plus control units at the Advanced BiofuelsProcess Demonstration Unit (ABPDU, Lawrence Berkeley NationalLaboratory, Emeryville, Calif.). The seed strain is inoculated in 500 mlYPD in a 1-L flask and grown overnight to an OD of 4.3. Each bioreactoris loaded with 900 ml YPD, 100 ml inoculum, and 200 ml dodecane (spikedwith internal standard). Temperature is maintained at 30° C. via waterflow through the reactor jacket. The pH is maintained at 5.0 byauto-dispensing a base solution (2N NH₄OH). Dissolved oxygen ismaintained at 20% via stirring from 400 to 800 rpm (primary cascade),then sparging air from 1 vvm (volume of air per volume of liquid perminute) to 1.5 vvm (secondary cascade). The feed contained 500 g/Lglucose, 10 g/L yeast extract, 10 g/L (NH₄)₂SO₄, 8 g/L KH₂PO₄, 4 g/LMgSO₄, 0.8 g/L NaCl, and 0.5 g/L CaCl₂ and is added as described in theresults section.

GC-MS and Extracellular Metabolite Analysis

Samples for GC-MS analysis of fatty alcohol content are analyzed on anAgilent 7890A GC equipped with an Agilent 5975 MS detector and anAgilent DB-5MS column. The inlet is set to 300° C., flow at 1 mL/min;the oven to 150° C. for 2 min, then ramped at 30° C./min to 250° C., andheld for 2 min. The solvent delay is set to 4 min (or as required toavoid the dodecane peak). An authentic hexadecanol standard (Sigmacatalog W255408) is used to determine titer.

Extracellular metabolites are analyzed on an automated photometricGallery Analyzer following the manufacturer's instructions.Preparation of Biomass Hydrolysates using Renewable Liquids

Switchgrass (Panicum virgatum) and sorghum (Sorghum bicolor) are kindlyprovided by Idaho National Laboratory. The air-dried biomass is milledusing a 40-mesh screen, sieved to the nominal sizes of 40-60 mesh(250-400 μm), and air-dried until the moisture is <10%. The resultingbiomass is converted into fermentable sugars in a one-pot, two-stepprocess consisting of biomass pretreatment using cholinium-based ILfollowed by enzymatic hydrolysis, modified from a previously reportedprocedure (Xu et al., 2016). Briefly, 20 g dry biomass is mixed with anIL solution containing 20 g IL and 160 g water. The ILs used arecholinium lysinate, cholinium alpha-ketoglutarate, or choliniumaspartate. The 200-g mixture is thoroughly mixed and loaded in a 500-mLParr reactor and heated to 140° C. for 3 hrs. The reactor is then cooledto room temperature using cooling water, and all the pretreated biomassslurry transferred to a filter membrane for solid-liquid separation. Thesolid fraction is collected, mixed with distilled water, and pH-adjustedto 5 with HCl. The weight of the mixture after pH adjustment is adjustedto 200 g with distilled water, and A the content transferred into a 14,shake flask for saccharification. The saccharification is carried out at50° C. and pH 5 using Novozymes enzyme mixtures Cellic® CTec2 and HTec2,with an enzyme dosage of 20 mg protein per grain glucan and 2 mg proteinper gram xylan, respectively. The resulting slurry is filtered using a0.2-um filter and used to dissolve 10 g/L yeast extract and 20-g/Lpeptone, making yeast extract-peptone-biomass hydrolysate media. In theresults, YPBiomass refers to sorghum treated with cholinium aspartate.

Microscopy

Strains for microscopy are grown in 5 ml YPD overnight, thenback-diluted 1:100into the same medium and grown 3-6 hrs at 200 rpm and30° C. Then, 1 ml of culture volume is centrifuged at 10,000×g on atable-top centrifuge, washed with 1× water, and imaged using a Zeiss LSM710 confocal system mounted on a Zeiss inverted microscope with a 63×objective and processed using Zeiss Zen software.

Results and Discussion Quantitative Analysis of Metabolic Flux andGlobal Protein Levels in First-Generation Fatty Chemical Producer

This work began with a quantitative analysis of metabolic fluxes andglobal protein levels to characterize this first-generation fattychemical producer strain and identify possible strategies to improveproduction. In a previous report, the native promoters driving the threefatty acid biosynthetic genes ACC1, FAS1, and FAS2 was replaced with thestrong TEF1 promoter and added terminal enzymes to produce ˜100 mg/Lfatty alcohols (using a FAR from Mus musculus) and ˜400 mg/L free fattyacids (using the thioesterase TesA from Escherichia coli, and deletingthe fatty acyl-CoA ligases encoded by FAA1 and FAA4) (Runguphan andKeasling, 2013). In this study, TesA is chromosomally integrated,expressing it from the TEF1 promoter, and analyzed the resulting strain,yL401, using metabolic flux analysis and global proteomics to identifybottlenecks in the biosynthetic pathway. Modeling metabolic fluxesallowed us to identify how substrates and cofactors involved in fattychemical production are used and produced by other cellular processesand thus identify possible targets for genetic modification. Layeringglobal protein expression data on top of the flux analysis allowed us toidentify genes whose differential expression may provide clues intocellular responses to the fatty chemical pathway, as well as a check onexpression levels of enzymes in the fatty acid biosynthetic pathway, oralong metabolic routes one may wish to engineer.

To quantify global protein expression, shotgun proteomics on yL401 isperformed as well as its un-engineered parent, BY4741. For both strains,many of the most highly expressed proteins—detected at between ˜0.5% and˜1.4% of total soluble protein—are glycolytic enzymes (Eno2p, Tdh3p,Tdh2p, Eno1p, Pgk1p), heat shock proteins (Ssa1p, Ssa2p, Ssb1 p, Ssb2p),translation elongation factors (Yef3p and Tef1p), and alcoholdehydrogenase 1 (Adh1p). As expected, proteins with increased expressionin yL401 included the three fatty acid biosynthetic enzymes Acc1p,Fas1p, and Fas2p (expressed chromosomally from the TEF1 promoter).Neither FAS subunit is detectable in BY4741, yet both reached ˜0.25% ofsoluble protein in yL401. Acc1p is also undetectable in BY4741, but inyL401 reached only ˜0.02% of soluble protein. Strain yL401 also showedincreased expression of several ribosomal and translation-initiationproteins (including Rp13p, Cdc33p, Mnp1p, and Rp139p), all induced 2-7fold relative to BY4741 but remaining under 0.05% of total solubleprotein. Proteins with decreased expression in yL401 included severalamino acyl-tRNA ligases (Grs1p, Gln4p) and interestingly, theretrotransposon Tyl Gag-Pol protein (which dropped by half from 0.17% ofsoluble protein in BY4741). Proteins expressed at intermediate levels inboth strains included the cytosolic NADP-dependent acetaldehydedehydrogenase (Ald6p); the other four ALD isoenzymes—two are cytosolicand NAD-dependent, the other two mitochondrial—are either not detectedor found at nominal levels (less than 0.05% of total protein). Ofpentose phosphate pathway enzymes, glucose-6-phosphate dehydrogenase(Zwf1p), the first and rate-limiting enzyme in the pentose phosphatepathway, is not detected (Thomas et al., 1991), but did observe severalother enzymes involved in the pathway, e.g., Gnd1p. None of the enzymesinvolved in the tricarboxylic acid (TCA) cycle or glyoxylate cycle aredetected at appreciable levels. Of amino acid biosynthesis enzymes,glutamate dehydrogenase (GDH) isoenzyme 1 is the most highly expressedat 0.12% of total soluble protein.

To model the metabolic flux distribution in yL401, metabolite levels ismeasured and ¹³C labeling patterns and used the JBEI QuantitativeMetabolic Modeling library (García Martín et al., 2015) to model fluxthrough genome-scale model iMM904 (Mo et al., 2009), adding fatty acylthioesterase reactions (EC number 3.1.2.2). The reconstruction showscarbon from glucose mostly following glycolysis, then the pyruvatedehydrogenase (PDH) bypass from pyruvate to acetaldehyde, and lastly toethanol (FIG. 1). At high glucose levels, yeast primarily fermentscarbon via the PDH bypass rather than oxidize it via mitochondrialrespiration, a well-documented phenomenon known as the Crabtree Effect(Zampar et al., 2013). Branching from central carbon metabolism, thisreconstruction shows the metabolic route to this product: some of theacetaldehyde is converted to acetate, then acetyl-CoA, malonyl-CoA, andlastly fatty acyl-CoA and derived chemicals. From acetaldehyde, only˜16% of the carbon flux is converted to acetate; the rest to ethanol (byalcohol dehydrogenase, ADH). The production of ethanol is a mechanism torecycle the large flux of NADH produced by glyceraldehyde-3-phosphatedehydrogenase (GapDH), since respiratory consumption of NADH isrepressed. The other main non-respiratory mechanism to recycle NADH isto produce glycerol; ˜14% of carbon entering glycolysis is routed tothis side product. This NADH/NAD⁺ flux is independent of the othercofactor pool, NADPH/NADP⁺, which is used in several anabolic reactionsincluding fatty acid biosynthesis (two NADPH are consumed per twocarbons added to the growing fatty acid chain). Yeast maintain[NAD⁺]/[NADH]>>1, and [NADP⁺/NADPH]<1, such that the cofactorconcentration gradients favor reduction of NAD⁺ for NADH/NAD⁺-dependentreactions, and oxidation of NADPH for NADPH/NADP⁺-dependent reactions.This reconstruction shows most NADPH being produced by ALD (˜67%), withonly a small amount (˜25%) from the pentose phosphate pathway. The vastmajority of all NADPH is consumed by GDH (˜60%) and fatty acidbiosynthesis (˜40%). Altogether, on a basis of carbon entering fromglucose, ˜33% is routed to ethanol, ˜20% to CO₂, ˜14% to glycerol, ˜2%to acetate, ˜16% to biomass, and ˜15% to fatty acid product.

The proteomics and flux results allowed us to identify possiblebottlenecks or imbalances in the pathway. For example, that Ald6p is thepredominant ALD isoform means flux through this reaction producesNADPH—rather than NADH if another ALD isoenzyme are prevalent. Followingthe carbon flux to the product, on a basis of one acetyl-CoA, thestoichiometry is 0.5× Glucose+1.38× ATP+1.25× NADPH−1 NADH=0.125× C₁₆FOH(ADP, NADP⁺, NAD⁺, H⁺, and CO₂ not included for simplicity). Even thoughALD flux produces NADPH, the metabolic route from glucose to fattyalcohol requires additional NADPH and produces excess NADH, an imbalancesought to be remediate (discussed in a later section). First, however,increasing carbon flux into fatty alcohol production by pull-push-blockengineering is focused upon: “pulling” on the pathway by overexpressingthe terminal enzyme; “pushing” by overexpressing enzymes to overcomebottlenecks on the metabolic route to the product; and “blocking”unwanted consumption of products or intermediates by deleting genescatalyzing undesirable reactions (Tai and Stephanopoulos, 2013).

TABLE 1 Strains. The following strains are available from the JBEIregistry (webpage for: public-registry.jbei.org) Strain yL# Parent(additional genetic changes) Reference BY4741 7 MATa, his3Δ1, leu2Δ0,met15Δ0, ura3Δ0 Runguphan et al., 2013 WRY1 400 BY4741(acc1::P_(TEF1)-ACC1, fas1::P_(TEF1)-FAS1, Runguphan et al., 2013fas2::P_(TEF1)-FAS2) TesA 401 WRY1 (208a::P_(TEF1)-EcTesAcyt-T_(CYC1),faa1Δ, faa4Δ) MaFAR2 402 WRY1 (208a::P_(TEF1)-MaFAR2-T_(CYC1)) MaFAR7403 WRY1 (208a::P_(TEF1)-MaFAR7-T_(CYC1)) TaFAR1 404 WRY1(208a::P_(TEF1)-TaFAR1-T_(CYC1)) MmFAR1 405 WRY1(208a::P_(TEF1)-MmFAR1-T_(CYC1)) FAR 406 yL405(1622b::P_(GAL1)-MmFAR1-T_(TDH1)) FAR-GFP 407 yL405(1622b::P_(GAL1)-MmFAR1-GFP-T_(TDH1)) MBP-FAR 408 yL405(1622b::P_(GAL1)-MBP-MmFAR1-T_(TDH1)) MBP-FAR-GFP 409 yL405(1622b::P_(GAL1)-MBP-MmFAR1-GFP-T_(TDH1)) ACC1** 410 yL405(YPRCd15c::P_(GAL1)-ACC1**-T_(ENO2)) HFA1cyt 411 yL405(YPRCd15c::P_(GAL1)-HFA1cyt-T_(ENO2)) dga1Δ 412 yL404 (dga1Δ) lro1Δ 413yL404 (lro1Δ) are2Δ 414 yL404 (are2Δ) are1Δ 415 yL404 (are1Δ) pxa1Δ 416yL404 (pxa1Δ) pox1Δ 417 yL404 (pox1Δ) hfd1Δ 418 yL404 (hfd1Δ) adh6Δ 419yL404 (adh6Δ) tdh3Δ::BcGapN 420 yL404 (tdh3Δ::BcGapN) tdh3Δ::KlGapDH 421yL404 (tdh3Δ::KlGapDH) pgi1Δ::ZWF1 422 yL404 (pgi1Δ::ZWF1) gdh1Δ 423yL404 (gdh1Δ) OLE1 424 yL405 (1014a::P_(TEF2)-OLE1-T_(ADH1)) opi1Δ::INO2425 yL405 (opi1Δ::P_(TPI1)-INO2-T_(PGK1)) rpd3Δ 426 yL405 (rpd3Δ) 2RA427 yL406 (YPRCd15c::P_(GAL1)-ACC1**-T_(ENO2)) 2RAh 428 yL427 (hfd1Δ)2RAha 429 yL427 (hfd1Δ, adh6Δ) 2RAhag 430 yL427 (hfd1Δ, adh6Δ, gdh1Δ)3RAhag 431 yL430 (1114a::P_(GAL1)-MmFAR1-T_(TDH1)) 2RAhagd 432 yL430(dga1Δ) 2RAhagdO 433 yL430 (dga1Δ, 1014a::P_(TEF2)-OLE1-T_(ADH1))2RAhagdOG 434 yL433 (gal80Δ::P_(TDH3)-GAL4)Pull: Comparing Fatty acyl-CoA Reductase Variants

To pull flux towards the product, first the only heterologous enzyme inthe pathway is focused, fatty acid reductase (FAR), which converts fattyacyl-CoA produced by fatty acid biosynthesis into fatty alcohols.Besides an earlier report expressing a Mus musculus FAR in S. cerevisiae(Runguphan and Keasling, 2013), other labs have reported onheterologously expressing a FAR from Tyto alba (Feng et al., 2015) andtwo from Marinobacter hydrocarbonasticus (Liu et al., 2013; Wahlen etal., 2009; Willis et al., 2011). To determine the variant most effectivefor fatty alcohol production in S. cerevisiae, the four FAR codingsequences (MmFAR1, TaFAR1, MaFAR2, and MaFAR7 are codon-optimized for S.cerevisae) are chromosomally integrated individually into the fattyacyl-CoA-overproducing strain WRY1, and compared fatty alcoholproduction using GC-MS (FIGS. 2A and 2B). Of the four enzymes, MmFAR1led to the highest titer at ˜550 mg/L (mass of extracted fattyalcohols/aqueous culture volume). TaFAR1 produced two thirds of that,and the two Marinobacter FARs negligible levels. MmFAR1 produced mostlyC16 fatty alcohols (87% C16, 7% C18, and 3% each C12 and C14), similarto the distribution from TaFAR1. The strain containing thebest-performing MmFAR1 gene (208a::P_(TEF1)-FAR, acc1::P_(TEF1)-ACC1,fas1::P_(TEF1)-FAS1, fas2:: P_(TEF1)::FAS2) is the base strain for allsubsequent engineering and is henceforth referred to as yL405.

Having determined MmFAR1 to be the best enzyme candidate for fattyalcohol production, next it is overexpressed further to investigatewhether this reaction still limited production. Starting with yL405, asecond MmFAR is added—in a cassette containing the strongest promoter(from GAL1) and terminator (from TDH1) reported in recent studies (Leeet al., 2015; Reider Apel et al., 2016)—which increased fatty alcoholtiter by 16 fold (FIG. 3A). In parallel, yL405 a version of the secondMmFAR fused at the C-terminus to a green fluorescent protein (GFP) isadded to examine enzyme solubility and localization. Confocal microscopyshowed GFP localization in a pattern typical of the endoplasmicreticulum (ER) common in lipid metabolic enzymes (Natter et al., 2005)(FIG. 2B). This localization is not found to be problematic since manyyeast enzymes that utilize fatty acyl-CoA as substrate (e.g., fattyacyl-CoA elongase Elo1p; desaturase Ole1p; acyl-CoA:sterolacyltransferases Are2p and Are1p) localize to the ER; and since MmFar1pproduced high levels of fatty alcohols. However, since fatty acyl-CoA isproduced in the cytoplasm, it is wondered whether a soluble, cytoplasmicMmFar1p might access additional substrate and further increase titer.Since MmFar1p fused C-terminally to GFP maintained membranelocalization, it is hypothesized that the N-terminus might contain amembrane-targeting signal and that adding an N-terminal maltose bindingprotein (MBP) might result in a soluble fusion protein. Either MBP-FARor MBP-FAR-GFP (to verify localization) is introduced to yL405. However,the strain containing MBP-FAR-GFP did not show cytosolic GFPlocalization, and none of the fusion enzymes improved fatty alcoholproduction over an untagged second copy of MmFAR (FIG. 3A).

Push: Overexpressing acetyl-CoA Carboxylase Variants

To push flux through potential bottlenecks, the first committed—andlimiting (Tehlivets et al., 2007)—step of fatty acid biosynthesis isfocused upon: the production of malonyl-CoA by acetyl-CoA carboxylase(ACC), in yeast encoded by ACC1. Although in an earlier work a strongpromoter to drive ACC1 is introduced, the present shotgun proteomicsanalysis detected much lower levels of Acc1p compared to Fas1p and Fas2pexpressed from the same TEF1 promoter. It is known that the gene productof ACC1 is regulated post-transcriptionally, e.g., throughphosphorylation sites that inhibit ACC1 activity. Recent work has shownthat mutating two serine residues on Acc1p (S659A, 51157A) can producehigh levels of malonyl-CoA-derived products (Shi et al., 2014).Therefore yL405 is introduced into a second copy of ACC1 containingthese two mutations (ACC1**) expressed from the strong GAL1 promoter. Inparallel, a cytosolic version of S. cerevisiae's native mitochondrialACC is constructed, HFA1, by replacing the latter's N-terminalmitochondrial targeting signal with the ACC1 N-terminus (HFA1cyt). Thisfusion protein has been shown to rescue an acc1Δ phenotype (Hoja et al.,2004) and does not contain any Snf1p target sites, or presumably anyother uncharacterized or indirect inactivation mechanisms since Hfa1pactivity is required for proliferating mitochondria during respirationwhen Snf1p signaling is active. Both ACC proteins improved fatty alcoholproduction compared to yL405, with ACC1** leading to the bestimprovement at 2.6 fold.

Block: Inhibiting Reactions Fatty Alcohols and Intermediates

To block undesirable reactions, eight genes encoding enzymes thatconsume fatty acyl-CoA or fatty alcohols are deleted. These includedenzymes that produce triacylglycerides (TAGs) for storage (DGA1 andLRO1) or steryl esters (ARE2 and ARE1), the peroxisomal importer (PXA1),the first step of beta-oxidation (PDX1), and two that dehydrogenatefatty alcohols (HFD1 and ADH6). These deletions in parallel on the basestrain yL405 are performed and screened for fatty alcohol production. Ofthe resulting eight deletion strains, dga1Δ had the highest titer (at6.8-fold higher than parent yL405 strain), followed by hfd1Δ (at2.6-fold higher), then adh6Δ (at 1.5-fold higher). None of the otherdeletions led to any improvement in titer.

Optimizing NADPH/NADP+ and NADH/NAD+ Cofactor Usage

Besides pull-push-block engineering of carbon flux, balancing redoxcofactor usage is focused upon. As discussed above, the fatty alcoholpathway produces excess NADH and requires additional NADPH. To remediatethis redox cofactor imbalance in the fatty alcohol pathway, firstreplacing native NADH-producing GapDH with an NADPH-producing variant ashas been pursued for other bioproducts is considered (Guo et al., 2011;Kildegaard et al., 2016; Zhang et al., 2011). From the proteomicsresults, it is found that of the three GapDH isoenzymes in yeast, Tdh3pis the most highly expressed. Thus TDH3 is replaced with either anNADPH-producing GapDH from Kluyveromyces lactis (KlGapDH) (Verho et al.,2002), or a non-phosphorylating GapDH from Bacillus cereus (BcGap1V)(Guo et al., 2011) in the baseline yL405 strain. Neither resultingstrain produced more fatty alcohols than the parent (FIG. 3A). It isnoted that the reaction catalyzed by GapDH has a positive ΔG⁰=1.5kcal/mol (Stryer, 1988). Normally, a chemical driving force afforded bya high cellular [NAD⁺]/[NADH] ratio makes the concentration-adjusted ΔGspontaneous. For an NADP⁺-dependent reaction, however, the cofactorratio is inverted ([NADP⁺]/[NADPH]<1) suggesting such a reaction mightresult in a bottleneck, or futile cycling between a forward,NADH-generating flux catabolized by the remaining native Tdh1p andTdh2p, and a reverse, NADPH-consuming flux catabolized by KlGapdhp. Thereaction catalyzed by BcGapnp, on the other hand, has a very favorableΔG⁰, but skips the ATP-generating step that normally follows GapDH, thuscreating a pathway ATP deficit. Similar thermodynamic considerationsstemming from concentration gradients across the redox cofactor pairsare relevant to other pathways.

Next, it is sought to increase NADPH availability for fatty acidbiosynthesis. This flux reconstruction indicated that the fatty acidpathway consumes only ˜5% of cellular ATP, but 40% of cellular NADPH,suggesting the latter cofactor may limit production. The quantitativeanalysis further showed that (1) carbon flux through the NADPH-producingpentose phosphate pathway is only 2% of that through PGI, (2) Pgilp isexpressed highly, and (3) Zwflp—the first and limiting enzyme of thepentose phosphate—is undetectable even though downstream pentosephosphate enzymes are detected. Thus, it is attempted to force fluxthrough the pentose phosphate pathway by deleting PGI1 and in its placeoverexpressing ZWF1 (pgi1Δ::ZWF1), also in the yL405 strain. However,the resulting strain grew slowly and barely produced any fatty alcohols(FIG. 3A).

Lastly, it is attempted to minimize NADPH consumed by competingreactions. This flux analysis indicated that 60% of all NADPH isconsumed to produce glutamate (from alpha-ketoglutarate and ammonia)with the two isoenzymes catabolizing this reaction, Gdh1p and Gdh3p,expressed at 0.12% and 0.034% of soluble protein, respectively. It istheorized that deleting GDH1 might slow down glutamate biosynthesis andfree up NADPH for fatty acid biosynthesis, as well as carbon. It is thusintroduced a gdh1Δ deletion into yL405, resulting in a 2.7-foldimprovement in fatty alcohol production.

Perturbations to Fatty Acid Regulation

The fatty acid pathway is energy intensive and regulated at severallevels (Tehlivets et al., 2007). Some of the strategies address knownlevels of regulation—e.g., using a strong constitutive promoter tooverexpress ACC1, FAS1, and FAS2; or abolishing post-translationalphosphorylation sites on ACC11'yet additional known or unknownregulatory mechanisms may continue to limit pathway flux. A recent studyshowed that deleting a histone deacetylase encoded by RPD3 dramaticallyincreased fatty alcohol production from an S. cerevisiae strainexpressing TaFAR (Feng et al., 2015). In yL405, rpdΔ4 did not yield anyimprovement (FIG. 3A). The gene encoding the negative regulator of fattyacid biosynthesis, OPI1, is also deleted and in its place overexpressedthe positive regulator encoded by INO2 in yL405. However, theopi1Δ::INO2 replacement does not improve fatty alcohol titers (FIG. 3A).

Lastly, a report found that (1) a correlation in mammalian tissuesbetween lipid accumulation (as TAGs) and expression level ofΔ9-desaturase (which produces mono-unsaturated fatty acyl-CoA), and (2)that overexpression of Δ9-desaturase Yarrowia lipolitica led todramatically increased levels of TAGs in an engineered strain (Qiao etal., 2015). To explore the possibility of Δ9-desaturase increasing fluxthrough the fatty alcohol pathway in S. cerevisiae—by increasingmembrane fluidity and access of MmFar1p to substrate (Degreif et al.,2017), or through an indirect feedback inhibition as in other organisms(Zhang et al., 2012)—the native S. cerevisiae Δ9-desaturase (encoded byOLE1) in yL405 is overexpressed, resulting in a 4-fold improvement infatty alcohol titer (FIG. 3A).

Combining Beneficial Genetic Edits into Highest-Producing Strain

Having found several genetic modifications that improved fatty alcoholtiters, beneficial changes into a high-producing strain are nextstacked. Starting with the best single-edit strain containing a secondcopy of FAR, then ACC1 ** is added, resulting in an additional 4.6-foldimprovement over yL405 (FIG. 3B). Then deletions of hfd1Δ, adh6Δ, andgdh1Δ are stacked, increasing titer up to 32.7-fold above yL405. Becausea second copy of FAR led to the most dramatic improvement in titer inthe single-gene edit strains, a third copy is added to examine whetherFAR activity remained limiting, but found the resulting strain producedless than parent (FIG. 3B). Instead, the remaining beneficialsingle-gene edits dga1Δ and OLE1 are combined to reach a titer 40-foldhigher than yL405.

Lastly, it is sought to deregulate the GAL1 promoter driving MmFAR andACC1 ** by deleting the negative regulator of galactose metabolism(GAL80) and in its place introducing a copy of the positive regulator(GAL4). This perturbation in galactose metabolism has the advantage ofmaking the GAL1 promoter constitutively active in glucose, allowing usto achieve high production using this inexpensive sugar as the carbonsource. The final yL427 strain, containing two copies of MmFAR, ACC1 **,deletions of dga1Δ, hfd1Δ, adh6Δ, and gdh1Δ, overexpression of OLE1, anda gal80Δ::GAL4 replacement produced 43-fold more fatty alcohols than theoriginal yL405 strain (FIG. 3B). At this level of production,precipitation of fatty alcohols from the saturated dodecane overlay inyL434 cultures is observed (FIG. 3C).

Fatty alcohol production from biomass sugars released using renewableionic liquid.

Having demonstrated a high-producing strain, then production fromlignocellulosic feedstocks derived from non-food bioenergy crops isevaluated. Specific combinations of anions and cations that are liquidsat room temperature (termed “ionic liquids”, ILs) have been shown todeconstruct plant biomass, allowing for subsequent enzymaticde-polymerization to free constituent sugars (Zhu et al., 2006).However, these processes are limited by high costs of conventional ILs,water usage, and toxicity. Recently, attention has shifted to ILsderived from benign cholinium cations stabilized with readily availablebio-based anions (Petkovic et al., 2010; Sun et al., 2016). A recentlydemonstrated one-pot process involving biomass pretreatment with ILscomposed of cholinium and amino acids followed by enzymaticsaccharification requires minimal water and unit operations, thusdramatically lowering process costs (Xu et al., 2016).

To examine the feasibility of using lignocellulosic feedstocks toproduce fatty alcohols, two types of non-food crops are pretreated(switchgrass and sorghum) with cholinium-based ILs containing threecommon bio-based anions (lysinate, alpha-ketoglutarate, or aspartate) ina one-pot process (see Materials and Methods). The six resulting biomasshydrolysate media all contained ˜1% glucose, and all produced more than150 mg/L fatty alcohols using the baseline strain yL405. The biomasshydrolysates media derived from sorghum pretreated with choliniumaspartate produced the most at 400 mg/L, fatty alcohols, and is furtherreferred to as YPBiomass. Then the highest producing yL434 strain inYPBiomass is grown (as well as traditional rich media YPD and YPG) inbaffled flasks, finding in preliminary experiments that greater aerationlead to improved production. Fatty alcohol levels reached 0.7 g/L inYPBiomass, corresponding to ˜0.06 g/g-glucose, already above the highestreported titers and yields (in shake flasks) using any media (Zhou etal., 2016). In YPD medium, fatty alcohol production reached 1.2 g/L and0.07 g/g-glucose (FIGS. 4A to 4C), corresponding to ˜20% of the maximumtheoretical yield, well above all previous reports of fatty alcoholproduction in S. cerevisiae.

Fed-Batch Bioreactor Fermentations

Finally, maximizing titer and demonstrating scale-up are realized infed-batch fermentation. Previous work on S. cerevisiae has suggestedmaximal yields depend on a balance between feeding enough sugar tomaintain high product flux, but not too much as to induce overflowmetabolism (Mazzoleni et al., 2015). To that end, 2-L scale bioreactorsare set up with feed strategies aimed at minimizing overflow metabolismand maximizing titer. For all four bioreactors, yL434 is cultured in aninitial 1-L YPD in batch operation until glucose depletion (as indicatedby O₂ spike), and then added concentrated glucose following differentfeed strategies: bioreactor A contained half the normal glucoseconcentration in batch phase (1% vs. 2% for others) and is fed aconstant ˜1.5 g/hr concentrated glucose; bioreactor B is pulse-fed ˜1 g/hr (as ˜1 g pulses of concentrated glucose upon carbon source depletionas indicated by O₂ spike, occurring every ˜1 hr); bioreactor C aconstant ˜3 g/hr; and bioreactor D a constant ˜1.5 g/hr. All fourbioreactors reached ODs of 25-31 and fatty alcohol titers >3 g/L and arestill climbing at the end of the allotted 9-day fermentation time (FIGS.5A to 6D). Bioreactor A showed the greatest rate of fatty alcoholproduction in fed-batch phase, the highest final titer, and the lowestfeed consumption, with the overlay showing extensive precipitation offatty alcohols. The final titer is 6 g/L, and the yield 58 mg/g-glucose,corresponding to 17% of the maximum theoretical yield.

Conclusions

In this work, S. cerevisiae is engineered for high-level production offatty alcohols guided by quantitative analysis of global proteinexpression and flux modeling. Four heterologous fatty acid reductasesare compared, finding high activity and ER localization from a Musmusculus FAR. After screening an additional 21 single-gene edits, thefollowing successful strategies to improve titer are identified: (1)increasing expression of MmFAR1, (2) deleting competing reactionsencoded by DGA1, HFD1, and ADH6, (3) overexpressing a mutant of thebottleneck enzyme encoded by ACC1 insensitive to post-transcriptionaland post-translational repression, (4) limiting NADPH and carbon flux toglutamate biosynthesis by deleting the enzyme encoded by GDH1, and (5)limiting fatty acid pathway repression by overexpressing the desaturaseencoded by OLE1. The final strain containing eleven geneticmodifications compared to the parent BY4741 strain produced 1.2 g/Lfatty alcohols in shake flasks.

High-level production from feedstocks produced from non-food crops andcholinium-based renewable bionic ILs is also demonstrated, reaching atiter of 0.7 g/L in shake flasks. Lastly, scale-up fermentation andexplored alternative feeding strategies aimed at limiting overflowmetabolism is demonstrated, achieving a titer of 6.0 g/L in a 2-L,fed-batch bioreactor. These titers are the highest for fatty alcoholsreported to date for S. cerevisiae. To our knowledge, this is also thefirst report of a bioproduct produced by yeast from feedstocks derivedsolely from biomass.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A genetically modified yeast cell comprising atleast six or more of the following modifications: (a) an increasedexpression of Mus musculus fatty acid reductase (FAR) (MmFAR1), orfunctional fragment thereof; (b) an increased expression of acetyl-CoAcarboxylase (ACC1), or functional fragment thereof: (c) an increasedexpression of fatty acid synthase 1 (FAS1), or functional fragmentthereof: (d) an increased expression of fatty acid synthase 2 (FAS2), orfunctional fragment thereof: (e) a reduced expression of or knocked outfor DGA1; (f) a reduced expression of or knocked out for HFD1; (g) areduced expression of or knocked out for ADH6; (h) an increasedexpression of a mutant of the bottleneck enzyme encoded by ACC1insensitive to post-transcriptional and post-translational repression,or functional fragment thereof; (i) a reduced expression of or knockedout for GDH1; and (j) an increased expression of the desaturase encodedby OLE1, or functional fragment thereof.
 2. A method of constructing thegenetically modified yeast cell of the present invention comprising atleast six or more of the following steps: (a) introducing a firstnucleic acid encoding MmFAR1, or functional fragment thereof,operatively linked to a promoter capable of expressing the MmFAR1 geneproduct in the yeast cell; (b) introducing a second nucleic acidencoding ACC1, or functional fragment thereof, operatively linked to apromoter capable of expressing the ACC1 gene product in the yeast cell,or replacing the native promoter of ACC1 with a promoter with a highertranscription activity, such as promoter TEF1; (c) introducing a thirdnucleic acid encoding FAS1, or functional fragment thereof, operativelylinked to a promoter capable of expressing the FAS1 gene product in theyeast cell, or replacing the native promoter of FAS1 with a promoterwith a higher transcription activity, such as promoter TEF1; (d)introducing a fourth nucleic acid encoding FAS2, or functional fragmentthereof, operatively linked to a promoter capable of expressing the FAS2gene product in the yeast cell, or replacing the native promoter of FAS2with a promoter with a higher transcription activity, such as promoterTEF1; (e) removing all or a portion of the DGA1 gene, or introducing anucleic acid into the yeast cell targeting the DGA1 gene or its geneproduct, such that the yeast cell has a reduced amount of activity ofthe DGA1 gene product; (f) removing all or a portion of the HFD1 gene,or introducing a nucleic acid into the yeast cell targeting the HFD1gene or its gene product, such that the yeast cell has a reduced amountof activity of the HFD1 gene product; (g) removing all or a portion ofthe ADH6 gene, or introducing a nucleic acid into the yeast celltargeting the ADH6 gene or its gene product, such that the yeast cellhas a reduced amount of activity of the ADH6 gene product; (h)introducing a fifth nucleic acid encoding a mutant of the bottleneckenzyme encoded by ACC1 insensitive to post-transcriptional andpost-translational repression, or functional fragment thereof,operatively linked to a promoter capable of expressing the mutant ACC1gene product in the yeast cell; (i) removing all or a portion of theGDH1 gene, or introducing a nucleic acid into the yeast cell targetingthe GDH1 gene or its gene product, such that the yeast cell has areduced amount of activity of the GDH1 gene product; (j) introducing asixth nucleic acid encoding OLE1, or functional fragment thereof,operatively linked to a promoter capable of expressing the OLE1 geneproduct in the yeast cell; and (k) introducing a seventh nucleic acidencoding a second copy of MmFAR1, or functional fragment thereof,operatively linked to a promoter capable of expressing the MmFAR1 geneproduct in the yeast cell.
 3. A method of producing a fatty alcohol froma genetically modified yeast cell comprising: (a) providing agenetically modified yeast cell of claim 1, and (b) growing or culturingthe genetically modified yeast cell in a medium such that thegenetically modified yeast cell produces one or more fatty alcohols, ora mixture thereof.